Methods for Expansion and Analysis of Cultured Hematopoietic Stem Cells

ABSTRACT

Methods and kits for propagating hematopoietic stem cells are provided. The methods comprise culturing cells in medium comprising one or more angiopoietin-like proteins, under conditions sufficient for expansion of HSCs. Angiopoietin-like proteins include angiopoietin-like protein 2, angiopoietin-like protein 3, angiopoietin-like protein 4, angiopoietin-like protein 5, angiopoietin-like protein 7, and microfibrillar-associated glycoprotein (Mfap4). Methods for identifying hematopoietic stem cells are provided and isolated hematopoietic stem cells are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/853,677, filed Aug. 10, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/438,847, filed May 23, 2006, now U.S. Pat. No.7,807,464, issued on Oct. 5, 2010, which claims the benefit of andpriority to U.S. Provisional Patent Application Ser. No. 60/753,212filed on Dec. 22, 2005 and U.S. Provisional Patent Application Ser. No.60/684,147 filed on May 24, 2005, the teachings of all of which areincorporated herein in their entireties.

GOVERNMENT FUNDING

This invention was made with support from the United States governmentunder grant numbers R01 DK 067356-01 and 075/P-IRFT, awarded by theNational Institutes of Health, and the United States government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The hematopoietic stem cell (HSC) through proliferation anddifferentiation gives rise to all of the cells in the hematopoieticsystem. Pluripotent HSCs are considered to be ideal candidates fordisease therapy and they serve as attractive target cells for deliveryof genes and gene products. It is desirable to have access to largeamounts of such HSCs; unfortunately, hematopoietic stem cells arepresent in extremely low numbers in tissues where they are found, suchas bone marrow and cord blood. Therefore, there is a need for improvedmethods of ex vivo cell culture systems capable of expandinghematopoietic cells, while maintaining stem cell pluripotency. It isalso important to be able to readily identify cells retaining suchpluripotency.

Difficulties in ex vivo expansion of HSCs have greatly hampered theirclinical utility as well as studies of their biological properties. Theidentification of new protein factors that can stimulate theself-renewal or prevent their apoptosis is an essential way to increasethe number of HSCs, including long term HSCs (LT-HSCs) in culture.

SUMMARY

As described herein, angiopoietin-like proteins promote the expansion ofHSCs. As a result of the present invention, hematopoietic stem cells canbe expanded to greater numbers in vitro.

The present invention includes methods of propagating hematopoietic stemcells in vitro comprising culturing one or more cells in culture mediumcomprising an angiopoietin-like protein, wherein at least one of thecells is capable of differentiating into one or more blood cell types.In an embodiment, a population of cells that contains stem cells iscultured in a medium that contains an effective amount of one or moreangiopoietin-like proteins under conditions sufficient for expansion ofthe cells. In a more particular embodiment, the method of propagatinghematopoietic stem cells comprises culturing one or more primary cellsfor at least five days in a serum free culture medium comprising anangiopoietin-like protein. The angiopoietin-like protein can be, forexample, angiopoietin-like protein 2, angiopoietin-like protein 3,angiopoietin-like protein 4, angiopoietin-like protein 5,angiopoietin-like protein 7, or Mfap4. In another embodiment, the methodof propagating hematopoietic stem cells comprises culturing at least oneprimary cell for at least five days in a serum free culture mediumcomprising an angiopoietin-like protein selected from the groupconsisting of angiopoietin-like protein 2, angiopoietin-like protein 3,angiopoietin-like protein 4, angiopoietin-like protein 5,angiopoietin-like protein 7, or Mfap4, wherein at least one of theprimary cells is capable of differentiating into one or more blood celltypes. In still another embodiment, the method of propagatinghematopoietic stem cells comprises culturing at least one human cell forat least five days in a serum free culture medium comprisingangiopoietin-like protein 5, wherein at least one of the human cells iscapable of differentiating into one or more blood cell types.

Cultured stem cells of the invention include hematopoietic stem cells,endothelial progenitor cells, bone marrow stromal stem cells,mesenchymal stem cells, embryonic stem (ES) cells and skeletal musclestem cells. Hematopoietic stem cells are particularly preferred.

HSCs can be cultured from any cell or population of cells which containsor has the potential to develop into HSCs. The population of cells cancontain at least 0.1% hematopoietic stem cells. In an embodiment, theone or more cells are primary cells. Typically, primary cells areobtained directly from tissue. Methods of obtaining primary cells arewell known in the art.

In another embodiment, the one or more cells can comprise total bonemarrow, umbilical cord blood cells, mobilized peripheral blood stemcells, or fetal liver cells. In another embodiment, the one or morecells comprises side population (SP) cells. In still another embodiment,the one or more cells comprises an isolated population of SP Sca1⁺ CD45⁺bone marrow cells. In other embodiments, the population of cellscultured can comprise CD34⁺ umbilical cord blood cells, AC133⁻(prominin) umbilical cord blood cells, CD34⁺ mobilized peripheral bloodstem cells, or AC133⁺ mobilized peripheral blood stem cells. In stillanother embodiment, the one or more cells can be human bone marrow CD34⁺and AC133⁺ cells. Other enriched populations of hematopoietic stem cellscan be used. Embryonic stem cells can also be used.

The one or more cells can be cultured for at least five days. In otherembodiments, the one or more cells can be cultured for at least 10 days,or for at least 2 weeks, or for at least four weeks.

The culture medium used for culturing the cells can comprise serum-freemedium. The culture medium can comprise an effective amount of one ormore additional factor(s), such as a cytokine(s). Suitable factorsinclude insulin-like growth factor (IGF) and at least one of fibroblastgrowth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF),under conditions sufficient for expansion of the cells. In certainembodiments, at least two of the latter factors are included. In furtherembodiments, at least three of the latter factors are included.

In another embodiment, the expanded HSCs retain at least some of thepluripotency of the initial stem cells or HSCs. Pluripotency includesstem cell activity or potential, such as the ability to differentiateinto other blood cell types or the ability to multiply withoutdifferentiating.

In another embodiment, the methods for promoting the expansion ofhematopoietic stem cells further comprises the step of selecting cellsafter culture (e.g., cultured cells) that express at least one positivecell surface marker selected from the group consisting of Sca-1⁺,IGF2-hFC⁺, CD31⁺, and Kit⁺ and/or do not express at least one negativecell surface marker selected from the group consisting of PrP, Lin, andCD62L. In another aspect, the cultured cells express at least twopositive cell surface markers and do not express at least two negativecell surface markers. In another aspect, the cultured cells express atleast three positive cell surface markers and do not express at leastthree negative cell surface markers.

In another embodiment, the cultured cells express Sca-1⁺, IGF2-hFC⁺,CD31⁺ and Kit⁺ and do not express PrP, Lin, and CD62L. In anotherembodiment, the cultured cells express Sca-1⁻ and IGF2-hFC⁺ and do notexpress PrP, Lin, and CD62L. In still another embodiment, the method forpromoting the expansion of hematopoietic stem cells comprises the stepof selecting cells after culture which specifically bind to anangiopoietin-like protein.

An embodiment of the invention provides methods for promoting theexpansion of hematopoietic stem cells in an individual in need of anexpanded number of hematopoietic cells, comprising administering to theindividual an angiopoietin-like protein in an amount effective topromote expansion of hematopoietic stem cells, thereby promotingexpansion of hematopoietic cells in the individual. In an embodiment forpromoting the expansion of hematopoietic stem cells in an individual,administration is by infusion.

In an embodiment, the individual is in need of expanded numbers ofhematopoietic cells due to the presence of a condition including but notlimited to reduced hematopoietic function, reduced immune function,reduced neutrophil count, reduced neutrophil mobilization, mobilizationof peripheral blood progenitor cells, sepsis, severe chronicneutropenia, bone marrow transplants, infectious diseases, leucopenia,thrombocytopenia, anemia, enhancing engraftment of bone marrow duringtransplantation, enhancing bone marrow recovery in treatment ofradiation, chemical or chemotherapeutic induced bone marrow aplasia ormyelosuppression, and acquired immune deficiency syndrome.

The present invention also provides methods of enhancing hematopoieticrecovery in a mammal that has undergone chemotherapy, comprisingadministering to the mammal an amount of an angiopoietin-like proteineffective to promote expansion of hematopoietic stem cells in themammal, thereby enhancing the hematopoietic recovery in the mammal thathas undergone chemotherapy.

The present invention is drawn to methods of administering hematopoieticstem cells to an individual. In an embodiment, the method comprisesobtaining hematopoietic stem cells from the individual or from a donor,culturing the cells in a culture medium comprising an angiopoietin-likeprotein, and transplanting the cultured cells into the individual.

The present invention also provides methods of treating an individual inneed of a hematopoietic stem cell-based therapy, comprising removinghematopoietic stem cells from the individual or from a donor; culturingthe cells in a culture medium containing an amount of anangiopoietin-like protein effective to promote expansion ofhematopoietic stem cells, harvesting the cultured cells, andtransplanting the cultured cells into the individual.

The present invention also provides methods of gene delivery andexpression in an individual, comprising removing hematopoietic stemcells from the individual or from a donor, culturing the cells in aculture medium containing an amount of an angiopoietin-like proteineffective to promote expansion of hematopoietic stem cells, introducingDNA into the cultured cells, harvesting the cultured cells, andtransplanting the cultured cells into the individual.

Another embodiment of the invention provides methods for screening for areceptor for an angiopoietin-like protein, comprising screening alibrary of candidate proteins or a library of genes encoding candidateproteins to identify proteins which bind to the angiopoietin-likeprotein and/or promote the expansion of hematopoietic stem cells inculture, wherein a candidate protein that binds to angiopoietin-likeprotein is indicative of a receptor for angiopoietin-like protein.

The present invention also provides methods of identifying hematopoieticstem cells. In an embodiment, the method comprises screening for cellswhich bind to at least one angiopoietin-like protein. In anotherembodiment, the screening for hematopoietic stem cells further comprisesselecting cells expressing at least one positive cell surface markerand/or not expressing at least one negative cell surface marker asdescribed above.

The present invention also includes kits for identifying hematopoieticstem cells. The kit includes at least one detectably labeledangiopoietin-like protein. The angiopoietin-like protein can be labeledwith any suitable detectable label such as an epitope, a fluorescentdye, or a radioactive label. Suitable epitopes include FLAG tags, myctags and other epitopes for which specific antibodies can be made or areavailable. Suitable fluorescent dyes include, for example, fluorescein,rhodamine, Cy3, and Cy5. Suitable radiolabels include, for example,¹²⁵I, ³²P, and ³⁵S. Methods for using detectable labels such as epitopetags, fluorescent dyes, and radiolabels are well known in the art.

Kits for propagating hematopoietic stem cells ex vivo (e.g., in vitro)are also provided. The kit can comprise medium suitable for culturingHSCs, one or more angiopoietin-like proteins, and optionally includesinstructions for expanding hematopoietic stem cells in vitro.

The various embodiments described herein can be complimentary and can becombined or used together in a manner understood by the skilled personin view of the teachings contained herein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 upper panel shows a schematic of the plasmid expressing the humanAngptl2 protein fused to a FLAG epitope at the C-terminus, and thebottom panel shows a Western blot of the 48 h conditioned medium of 293Tcells transfected by pcDNA3.1(−) (lane 1) or pcDNA3.1(−) encodingFLAG-Angptl2 (lane 2), probed with antibodies against FLAG epitope.

FIG. 2 shows micrographs of FACS sorted bone marrow SP Sca-1⁺ CD45⁺cells in various serum-free culture conditions, with and withoutAngptl2.

FIG. 3A is a bar graph showing bar percent repopulation 3 weeks, 4months, and 9 months after transplant with twenty freshly isolatedCD45.2 bone marrow SP Sca-1⁺ CD45⁺ cells cotransplanted with 1×10⁵CD45.1 competitors in the absence of prior culturing (bars 1, 6, and 11)or cultured as indicated.

FIG. 3B top panel shows representative FACS plots of peripheral bloodmononuclear cells from one mouse at 9 months post-transplant (cells frombar 15 of FIG. 3A) (the number in each quadrant is the percentage ofcells in each quadrant); the bottom panel shows the summary of data fromall mice in bars 13 and 15 of FIG. 3A.

FIG. 3C top panel shows representative FACS plots of peripheral bloodmononuclear cells from one mouse at 4 months after a secondarytransplantation; the bottom panel shows the summary of data from allmice at 4 months after a secondary transplantation of mouse bone marrowrepresented in bars 8 and 10 of FIG. 3A.

FIG. 4A shows a silver stain of 16 ng of purified E. coli-expressedAngptl2 (lane 2), and 2.5 ng of purified mammalian-expressedFLAG-Angptl2 protein (purified from an anti-FLAG column, lane 3)fractioned by SDS-PAGE.

FIG. 4B shows a Western blot of the purified Angptl2 (right lane) andcontrol (left lane) probed with anti-FLAG antibody.

FIG. 4C shows a Western blot of the purified Angptl2 (right lane) andcontrol BSA (left lane) probed with anti-Angptl2 mAb.

FIG. 5 is a bar graph showing that purified Angptl2 is a growth factorfor HSCs.

FIG. 6 left panel shows a Western blot of 48 hour conditioned medium of293T cells transfected by pcDNA3.1(−) (lane 1), FLAG-hAngptl2coiled-coil domain (lane 2), or FLAG-hAngptl2 fibrinogen-like domain(lane 3), probed with antibodies against hAngptl2; the right panel is abar graph showing percent repopulation 3 months and 6 months aftertransplant with twenty CD45.2 bone marrow SP Sca-1⁺ CD45⁻ cells culturedfor 5 d in serum-free mock-transfected 293T cell conditioned STIF medium(bars 1 and 6); in the same medium with 100 ng/ml E. coli-expressedfull-length Angptl2 (bars 2 and 7); and in the same medium with 100ng/ml mammalian-expressed full-length Angptl2 (bars 3 and 8).

FIG. 7A is a graph showing percent of mice repopulated with theindicated number of BM SP CD45⁺Sca-1⁺ cells.

FIG. 7B is a graph showing percent of mice repopulated with theindicated number of BM SP CD45⁺Sca-1⁺ cells after culturing for 10 d inserum-free conditioned STIF medium containing 100 ng/ml of purifiedAngptl2.

FIG. 7C is a graph showing percent of mice repopulated with theindicated number of BM SP CD45⁺Sca-1⁺ cells after culturing for 10 d inserum-free conditioned STIF medium containing 100 ng/ml of purifiedAngptl3.

FIG. 8 is a bar graph showing that purified Angptl3, Angptl5, andAngptl7 are growth factors for hematopoietic stem cells.

FIG. 9 is a bar graph showing that Angptl2-coiled coil domain, Angptl4,and Mfap4 expressed in 293T cell conditioned medium stimulated theproliferation of hematopoietic stem cells.

FIG. 10A left panel shows a silver stain of purifiedbacterially-expressed hAngptl7 (lane 2), purified wheat germ in vitrotranscribed GST-hAngptl5 (lane 3), purified mammalian-expressed hAngptl4(lane 4), and purified sf21-expressed mAngptl3 (lane 5) fractioned bySDS-PAGE; the right panel shows a bar graph of percent repopulation 4weeks, 3 months, or 6 months after transplant with twenty CD45.2 bonemarrow SP Sca-1⁺ CD45⁺ cells cultured for 5 d in serum-freeunconditioned STIF medium (bars 1, 6, and 11), or cultured in the samemedium with 100 ng/ml purified insect-expressed mAngptl3 (bars 2, 7, and12), or cultured in the same medium with 100 ng/ml purified hAngptl4(bars 3, 8, and 13), or cultured in the same medium with 100 ng/mlpurified GST-hAngptl5 (bars 4, 9, and 14), or cultured in the samemedium with 1 μg/ml purified bacterially-expressed hAngptl7 (bars 5, 10,and 15).

FIG. 10B left panel shows a Western blot of 48 hour conditioned mediumof 293T cells transfected by pcDNA3.1(−) (lane 1), pcDNA3.1 encodingFLAG-hMfap4 (lane 2), or pcDNA3.1 encoding FLAG-hFgl1 (lane 3), andprobed with an antibody specific for the FLAG epitope; the right panelshows a bar graph of percent repopulation 3 months or 6 months aftertransplant with twenty CD45.2 bone marrow SP Sca-1⁺ CD45⁺ cells culturedfor 5 d in serum-free mock transfected conditioned STIF medium (bars 1and 4), in conditioned STIF medium from 293T cells transfected bypcDNA3.1 encoding FLAG-hFgl1 (bars 2 and 5), or conditioned STIF mediumfrom 293T cells transfected by PCDNA3.1 encoding FLAG-hMfap4 (bars 3 and6).

FIG. 11A is a bar graph showing average relative expression of Angptl2(left panel) or Angptl3 (right panel) mRNA in cells having the indicatedphenotype (FL is fetal liver).

FIG. 11B is a bar graph showing average relative expression of Angptl2mRNA in adult mouse bone marrow hematopoietic cells and adult mouse bonemarrow cells having the indicated phenotype.

FIG. 11C is a bar graph showing average relative expression of Angptl3mRNA in adult mouse bone marrow hematopoietic cells and adult mouse bonemarrow having the indicated phenotype.

FIG. 12A is a bar graph showing percent repopulation 3 weeks or 4 monthsafter transplant with freshly isolated adult CD45.2 BM cells that eitherbind Angptl2 or not, as indicated; demonstrating that freshly isolatedHSCs bind Angptl2.

FIG. 12B is a bar graph showing percent repopulation 3 weeks or 3 monthsafter transplant with adult CD45.2 BM cells cultured in serum-freemedium with SCF, TPO, IGF-2, and FGF-1 for 4 d and that bind Angptl2 ornot, as indicated; demonstrating that 4-day cultured HSCs bind Angptl2.

FIG. 13 is a graph showing percent repopulation in NOD/SCID recipientmice two months after transplant with human cord blood cells culturedfor the indicated number of days in medium supplemented with theindicated growth factors.

FIG. 14 is a schematic diagram of hematopoietic stem cell self renewaland differentiation into different blood cell types.

FIG. 15 shows the amino acid sequences for exemplary angiopoietin-likeproteins.

DETAILED DESCRIPTION

As described herein, freshly isolated HSCs as well as cultured HSCs bindto angiopoietin-like proteins. In addition, as described herein,angiopoietin-like proteins promote the expansion of hematopoietic stemcells. The present invention is directed to hematopoietic stem cells,methods for propagating or expanding hematopoietic stem cells, andmethods of using the propagated hematopoietic stem cells.

Ex vivo Cultures of Hematopoietic Stem Cells

The present invention provides methods for promoting the expansion ofhematopoietic stem cells (HSCs) in culture (e.g., in vitro or ex vivo).The cell or cells to be cultured can include any cell that is capable ofdifferentiating into one or more blood cell types. Exemplary blood celltypes are shown in FIG. 14 and include phagocytic immune cells (e.g.,granulocytes), monocytes (e.g., macrophage precursor cells),macrophages, eosiniphils, erythrocytes, platelet forming cells (e.g.,megakaryocytes), T lymphocytes, B lymphocytes, and natural killer (NK)cells. Suitable cell(s) can also be capable of self renewal, that is,capable of propagating or increasing in number and remaining at the samedevelopmental stage as the parent cell.

Suitable cell(s) can be isolated, for example, from any known source ofhematopoietic stem cells, including, but not limited to, bone marrow,mobilized peripheral blood (MPB), fetal liver, and umbilical cord blood.Umbilical cord blood is discussed, for instance, in Issaragrishi et al.,N. Engl. J. Med. 332:367-369 (1995). Bone marrow cells can be obtainedfrom a source of bone marrow, including but not limited to, ilium (e.g.,from the hip bone via the iliac crest), tibia, femora, vertebrate, orother bone cavities. Other sources of stem cells include, but are notlimited to, ES cells, embryonic yolk sac, fetal liver, and fetal spleen.

The cell or cells can be subjected to methods of further enrichment forhematopoietic stem cells. Means for isolating hematopoietic stem cellsusing specific stem cell markers are known to those skilled in the art.

The one or more cells and the hematopoietic cells of the invention canbe derived from any suitable animal, e.g., human, non-human primates,porcine or murine. In one preferred embodiment, the cells are humancells.

For isolation of bone marrow, an appropriate solution can be used toflush the bone, including, but not limited to, salt solution, optionallysupplemented with fetal calf serum (FCS) or other naturally occurringfactors, in conjunction with an acceptable buffer. In an embodiment, thebuffer is at low concentration, generally from about 5 to about 25 mM.Convenient buffers include, but are not limited to, HEPES, phosphatebuffers and lactate buffers. Bone marrow can also be aspirated from thebone in accordance with conventional techniques.

Regarding lineage specific markers, the absence or low expression oflineage specific markers can be identified by the lack of binding ofantibodies specific to the lineage specific markers, useful in so-called“negative selection”. The source of cells for use in the methods of thepresent invention can be subjected to negative selection techniques toremove those cells that express lineage specific markers and retainthose cells which are lineage negative (“Lin⁻”). Lin⁻ generally refersto cells which lack markers such as those associated with T cells (suchas CD2, 3, 4 and 8), B cells (such as B220, CD48, CD10, 19 and 20),myeloid cells (such as Mac-1, Gr-1, CD14, 15, 16 and 33), natural killer(“NK”) cells (such as CD244, CD2, 16 and 56), RBC (such as Ter119, andglycophorin A), megakaryocytes (CD41), mast cells, eosinophils orbasophils. Methods of negative selection are known in the art. Lineagespecific markers also include CD38, HLA-DR and CD71. As used herein,“Lin⁻” refers to a cell population selected based on the lack ofexpression of at least one lineage specific marker.

Various techniques can be employed to separate the cells by initiallyremoving cells of dedicated lineage or having a particular phenotype.Monoclonal antibodies are particularly useful for identifying markersassociated with particular cell lineages, stages of differentiation, orparticular phenotypes. The antibodies can be attached to a solid supportto allow for crude separation. The separation techniques employed shouldmaximize the retention of viable cells in the fraction to be collected.Various techniques of different efficacy can be employed to obtain“relatively crude” separations. Such separations are up to 10%, usuallynot more than about 5%, of the total cells present not having the markercan remain with the cell population to be retained. In certainembodiments, not more than about 1% of the total cells present in thepopulation of retained cells do not have the marker. The particulartechnique employed will depend upon efficiency of separation, associatedcytotoxicity, ease and speed of performance, and necessity forsophisticated equipment and/or technical skill.

Procedures for separation can include, but are not limited to, physicalseparation, magnetic separation, using antibody-coated magnetic beads,affinity chromatography, cytotoxic agents joined to a monoclonalantibody or used in conjunction with a monoclonal antibody, including,but not limited to, complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g., plate, elutriation or anyother convenient technique. In certain embodiments, one uses a highthroughput technique to rapidly screen and separate different cells.

The use of physical separation techniques include, but are not limitedto, those based on differences in physical (density gradientcentrifugation and counter-flow centrifugal elutriation), cell surface(lectin and antibody affinity), and vital staining properties(mitochondria-binding dye rho123 and DNA-binding dye Hoechst 33342).These procedures are well known to those of skill in this art.

Techniques providing accurate and rapid separation include, but are notlimited to, flow cytometry (e.g., fluorescence activated cell sorting),which can have varying degrees of sophistication, e.g., a plurality ofcolor channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. Cells also can be selected by flowcytometry based on light scatter characteristics, where stem cells areselected based on low side scatter and low to medium forward scatterprofiles. Cytospin preparations show the enriched stem cells to have asize between mature lymphoid cells and mature granulocytes.

For example, in a first separation step, anti-CD34 can be labeled with afirst fluorochrome, while the antibodies for the various dedicatedlineages, can be conjugated to a fluorochrome with different anddistinguishable spectral characteristics from the first fluorochrome.While each of the lineages can be separated in more than one“separation” step, desirably the lineages are separated at the same timeas one is positively selecting for HSCs. The cells can be selected andisolated from dead cells, by employing dyes associated with dead cells(including but not limited to, propidium iodide (PI)). The particularorder of separation is not critical to this invention.

The cells obtained as described above can be used immediately or frozenat liquid nitrogen temperatures and stored for long periods of time,being thawed and capable of being reused. Once thawed, the cells can beexpanded by use of the methods described herein.

HSCs can be expanded by culturing one or more cells in an expansioncontainer and in a volume of a suitable medium. Cells populations highlyenriched in stem cells and methods for obtaining them are described inWO 95/05843; WO 95/03693 and WO 95/08105. In a preferred embodiment theone or more cells comprises a population of cells that is substantiallyenriched in hematopoietic stem cells. In another embodiment, thepopulation of cells is substantially free of stromal cells.

The cells can be cultured such that the culture well contains about1-100 cells per well. Where the population of cells is bone marrow, thecells can be cultured at a density of about 1×10² cells to about 1×10⁷cells/mL of medium. In another embodiment, the cells cells can becultured at a density of about 1×10⁵ cells to about 1×10⁶ cells/mL ofmedium. In another embodiment, the population of cells comprises SidePopulation (SP) bone marrow cells. The SP bone marrow cells can becultured at lower density, for example from about 1×10² to 5×10³cells/ml. In a separate aspect, the population of cells can be derivedfrom mobilized peripheral blood. The mobilized peripheral blood cellscan be cultured at a density of about 20,000 cells/mL to about 50,000cells/mL; in another embodiment, the mobilized peripheral blood cells iscultured at a density of about 50,000 cells/mL.

Any suitable expansion container, flask, or appropriate tube such as a12, 24 or 96 well plate, 12.5 cm² T flask or gas-permeable bag can beused in the method of this invention. Such culture containers arecommercially available from Falcon, Corning or Costar. As used herein,“expansion container” also is intended to include any chamber orcontainer for expanding cells whether or not free standing orincorporated into an expansion apparatus.

Various media can be used for the expansion of the stem cells includingDulbecco's MEM, IMDM, X-Vivo 15 (serum-depleted, Cambrex), RPMI-1640 andStemSpan (Stem Cell Technologies). In another embodiment, the cellculture medium is serum free. One serum free medium which can be used inthe methods of the invention is serum free StemSpan (Stem CellTechnologies). In another embodiment, the medium is supplemented with 10μg/ml heparin.

The media formulations for expansion of HSCs contain concentrations ofone or more angiopoietin-like proteins in a range from about 0.1 ng/mLto about 500 ng/mL. In another embodiment, from about 1 ng/mL to about100 ng/mL of one or more angiopoietin-like proteins is used. In anotherembodiment, from about 10 ng/ml to 50 ng/ml of one or moreangiopoietin-like proteins is used. Other useful concentrations ofangiopoietin-like proteins can be readily determined by one of ordinaryskill in the art using the teachings contained herein.

In another aspect, in addition to the angiopoietin-like protein of theinvention, the media formulations for expansion of HSCs are supplementedwith cytokines, including but not limited to fibroblast growth factor(FGF) (e.g. FGF-1 or FGF-2), insulin growth factor (e.g. IGF-2, orIGF-1), thrombopoietin (TPO), and stem cell factor (SCF). As indicatedabove, the concentrations of cytokines range from about 0.1 ng/mL toabout 500 ng/mL. In another embodiment, from about 1 ng/mL to about 200ng/mL of a cytokine is used. In another embodiment, from about 10 ng/mlto 100 ng/ml of a cytokine is used. In another embodiment, the cytokinesfor use in the invention are FGF-1, TPO, IGF-2, and SCF. In stillanother embodiment the SCF is present at 10 ng/ml concentration, TPO at20 ng/ml concentration, IGF-2 at 20 ng/ml concentration and FGF-1 at 10ng/ml concentration. Other cytokines may be added, alone or incombination, and include but are not limited to G-CSF, GM-CSF, IL-1α,and IL-11. Other useful concentrations of cytokines can be readilydetermined by one of ordinary skill in the art using the teachingscontained herein.

Transplantation

The expanded cultured hematopoietic stem cells of the invention can beused for a variety of applications, including transplantation, sometimesreferred to as cell-based therapies or cell replacement therapies, suchas bone marrow transplants, gene therapies, tissue engineering, and invitro organogenesis.

Hematopoietic progenitor cell expansion for bone marrow transplantationis a potential application of human bone marrow cultures. Humanautologous and allogeneic bone marrow transplantation are currently usedas therapies for diseases such as leukemia, lymphoma, and otherlife-threatening diseases. For these procedures, however, a large amountof donor bone marrow must be removed to ensure that there are enoughcells for engraftment. The methods of the present invention circumventthis problem. Methods of transplantation are known to those skilled inthe art.

Expanded hematopoietic stem cells are particularly suited forreconstituting hematopoietic cells in a subject or for providing cellpopulations enriched in desired hematopoietic cell types. This methodinvolves administering by standard means, such as intravenous infusionor mucosal injection, the expanded cultured cells to a patient.

The discovery that cells may be expanded ex vivo and administeredintravenously provides the means for systemic administration. Forexample, bone marrow-derived stem cells may be isolated with relativeease and the isolated cells may be cultured according to methods of thepresent invention to increase the number of cells available. Intravenousadministration also affords ease, convenience and comfort at higherlevels than other modes of administration. In certain applications,systemic administration by intravenous infusion is more effectiveoverall. In another embodiment, the stem cells are administered to anindividual by infusion into the superior mesenteric artery or celiacartery. The cells may also be delivered locally by irrigation down therecipient's airway or by direct injection into the mucosa of theintestine.

After isolating the cells, the cells can be cultured for a period oftime sufficient to allow them to expand to desired numbers, without anyloss of desired functional characteristics. For example cells can becultured from 1 day to over a year. In another embodiment, cells arecultured for 3 to 30 days. In another embodiment, cells are cultured for4 to 14 days. In yet another embodiment, cells can be cultured for atleast 7 days.

Between 10⁵ and 10¹³ cells per 100 kg person are administered perinfusion. In another embodiment, about 1×10⁸ to about 5×10¹² cells areinfused intravenously per 100 kg person. In another embodiment, betweenabout 1×10⁹ and 5×10¹¹ cells are infused intravenously per 100 kgperson. For example, dosages such as 4×10⁹ cells per 100 kg person and2×10¹¹ cells can be infused per 100 kg person.

In some embodiments, a single administration of cells is provided. Inother embodiments, multiple administrations are used. Multipleadministrations can be provided over periodic time periods such as aninitial treatment regime of 3 to 7 consecutive days, and then repeatedat other times.

With respect to cells as administered to a patient, an effective amountmay range from as few as several hundred or fewer to as many as severalmillion or more. In specific embodiments, an effective amount may rangefrom 10³ to 10⁸. It will be appreciated that the number of cells to beadministered will vary depending on the specifics of the disorder to betreated, including but not limited to size or total volume/surface areato be treated, as well as proximity of the site of administration to thelocation of the region to be treated, among other factors familiar tothe medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other complication commensurate with a reasonable benefit/risk ratio.As described in greater detail herein, pharmaceutically acceptablecarriers suitable for use in the present invention include liquids,semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds). Asused herein, the term biodegradable describes the ability of a materialto be broken down (e.g., degraded, eroded, dissolved) in vivo. The termincludes degradation in vivo with or without elimination (e.g., byresorption) from the body. The semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, e.g., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orby breakdown and elimination through natural pathways.

Several terms are used herein with respect to transplantation therapies,also known as cell-based therapies or cell replacement therapy. Theterms autologous transfer, autologous transplantation, autograft and thelike refer to treatments wherein the cell donor is also the recipient ofthe cell replacement therapy. The terms allogeneic transfer, allogeneictransplantation, allograft and the like refer to treatments wherein thecell donor is of the same species as the recipient of the cellreplacement therapy, but is not the same individual. A cell transfer inwhich the donor's cells have been histocompatibly matched with arecipient is sometimes referred to as a syngeneic transfer. The termsxenogeneic transfer, xenogeneic transplantation, xenograft and the likerefer to treatments wherein the cell donor is of a different speciesthan the recipient of the cell replacement therapy.

The expanded hematopoietic cells can be used for reconstituting the fullrange of hematopoietic cells in an immunocompromised host followingtherapies such as, but not limited to, radiation treatment andchemotherapy. Such therapies destroy hematopoietic cells eitherintentionally or as a side-effect of bone marrow transplantation or thetreatment of lymphomas, leukemias and other neoplastic conditions, e.g.,breast cancer.

Expanded hematopoietic cells are also useful as a source of cells forspecific hematopoietic lineages. The maturation, proliferation anddifferentiation of expanded hematopoietic cells into one or moreselected lineages may be effected through culturing the cells withappropriate factors including, but not limited to, erythropoietin (EPO),colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, SCF,interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, -13, etc., or withstromal cells or other cells which secrete factors responsible for stemcell regeneration, commitment, and differentiation.

Drug Discovery

Expanded hematopoietic cells of the invention are useful for identifyingculture conditions or biological modifiers such as growth factors whichpromote or inhibit such biological responses of stem cells asself-regeneration, proliferation, commitment, differentiation, andmaturation. In this way one may also identify, for example, receptorsfor these biological modifiers, agents which interfere with theinteraction of a biological modifier and its receptor, and polypeptides,antisense polynucleotides, small molecules, or environmental stimuliaffecting gene transcription or translation.

For example, the present invention makes it possible to preparerelatively large numbers of hematopoietic stem cells for use in assaysfor the differentiation of stem cells into various hematopoieticlineages. These assays may be readily adapted in order to identifysubstances such as factors which, for example, promote or inhibit stemcell self-regeneration, commitment, or differentiation.

The invention provides methods to identify receptors for theangiopoietin-like proteins. Receptors for these orphan ligands have notyet been identified.

Accordingly, the invention provides methods for screening for a receptorfor an angiopoietin-like protein, comprising screening a library ofcandidate proteins or a library of genes encoding candidate proteins toidentify proteins which bind to the angiopoietin-like protein and/orpromote the expansion of hematopoietic stem cells in culture. In anembodiment, a library of candidate genes is introduced into andexpressed in cells that do not normally bind to the angiopoietin-likeprotein of interest. These cells can then be exposed to one or moreangiopoietin-like proteins. Cells that bind to the angiopoietin-likeprotein and/or expand upon exposure to the angiopoietin-like protein areindicative of cells that contain an angiopoietin-like protein receptor.The cells that bind or expand in the presence of the angiopoietin-likeprotein can be isolated, and the transfected gene can be isolated fromthe cells.

Gene Cloning Strategies

One may also use the expanded cells of the invention to identify andclone genes whose expression is associated with proliferation,commitment, differentiation, and maturation of stem cells or otherhematopoietic cells, e.g., by subtractive hybridization or by expressioncloning using monoclonal antibodies specific for target antigensassociated with these biological events or characteristic of ahematopoietic cell type.

Gene Delivery and Expression

As described above hematopoietic stem cells are also important targetsfor gene delivery and expression in a subject. One such embodiment issometimes referred to as gene therapy.

According to the invention, the cultured expanded cells can be furthergenetically altered prior to reintroducing the cells into an individualfor gene therapy, to introduce a gene whose expression has therapeuticeffect on the individual. Methods for introducing genes into thecultured cells are provided in detail below.

In some aspects of the invention, individuals can be treated bysupplementing, augmenting and/or replacing defective and/or damagedcells with cells that express a therapeutic gene. The cells may bederived from cells of a normal matched donor or stem cells from theindividual to be treated (i.e., autologous). By introducing normal genesin expressible form, individuals suffering from such a deficiency can beprovided the means to compensate for genetic defects and eliminate,alleviate or reduce some or all of the symptoms.

Expression vectors may be introduced into and expressed in autologous orallogeneic expanded hematopoietic cells, or the genome of cells may bemodified by homologous or non-homologous recombination by methods knownin the art. In this way, one may correct genetic defects in anindividual or provide genetic capabilities naturally lacking in stemcells. For example, diseases including, but not limited to,β-thalassemia, sickle cell anemia, adenosine deaminase deficiency,recombinase deficiency, and recombinase regulatory gene deficiency maybe corrected in this fashion. Diseases not associated with hematopoieticcells may also be treated, e.g., diseases related to the lack ofsecreted proteins including, but not limited to hormones, enzymes, andgrowth factors. Inducible expression of a gene of interest under thecontrol of an appropriate regulatory initiation region will allowproduction (and secretion) of the protein in a fashion similar to thatin the cell which normally produces the protein in nature.

Similarly, one may express in expanded hematopoietic cells a ribozyme,antisense RNA or protein to inhibit the expression or activity of aparticular gene product. Drug resistance genes including, but notlimited to, the multiple drug resistance (MDR) gene, may also beintroduced into cells, e.g., to enable them to survive drug therapy. Forhematotrophic pathogens, such as HIV or HTLV-I, and HTLV II, the cellscan be genetically modified to produce an antisense RNA, ribozyme, orprotein which would prevent the proliferation of a pathogen inhematopoietic stem cells or differentiated cells arising from the stemcells. One may also disable or modulate the expression of a particulargenetic sequence by methods known in the art, including, but not limitedto, directly substituting, deleting, or adding DNA by homologousrecombination or indirectly by antisense sequences.

Transduction of Hematopoietic Stem Cell Cultures

The expanded hematopoietic stem cells of the invention can begenetically modified. The introduction of the gene into thehematopoietic stem cell can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors, adjuvant-assisted DNA, gene gun, catheters, etc. Inanother embodiment, a viral vector is used.

Polynucleotides are inserted into vector genomes using methods wellknown in the art. For example, insert and vector DNA can be contacted,under suitable conditions, with a restriction enzyme to createcomplementary ends on each molecule that can pair with each other and bejoined together with a ligase. Alternatively, synthetic nucleic acidlinkers can be ligated to the termini of restricted polynucleotide.These synthetic linkers contain nucleic acid sequences that correspondto a particular restriction site in the vector DNA. Additionally, anoligonucleotide containing a termination codon and an appropriaterestriction site can be ligated for insertion into a vector containing,for example, some or all of the following: a selectable marker gene,such as the neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and ColE1 for properepisomal replication; versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Other means are well known and available in the art.

Modification of hematopoietic stem cells can comprise the use of anexpression cassette created for either constitutive or inducibleexpression of the introduced transgene. Such an expression cassette caninclude regulatory elements such as a promoter, an initiation codon, astop codon, and a polyadenylation signal. It is necessary that theseelements be operable in the stem cells or in cells that arise from thestem cells after infusion into an individual. Moreover, it is necessarythat these elements be operably linked to the nucleotide sequence thatencodes the protein such that the nucleotide sequence can be expressedin the stem cells and thus the protein can be produced. Initiationcodons and stop codons are generally considered to be part of anucleotide sequence that encodes the protein.

A variety of promoters can be used for expression of the transgene.Promoters that can be used to express the gene are well known in theart. Promoters include cytomegalovirus (CMV) intermediate earlypromoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR,HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5promoter and the herpes simplex tk virus promoter. For example, one canuse a tissue specific promoter, i.e. a promoter that functions in sometissues but not in others. Such promoters include EF2 responsivepromoters, etc. Examples of promoters that may be used to causeexpression of the introduced sequence in specific cell types includeGranzyme A for expression in T-cells and NK cells, the CD34 promoter forexpression in stem and progenitor cells, the CD8 promoter for expressionin cytotoxic T-cells, and the CD11b promoter for expression in myeloidcells.

Regulatable promoters can be used. Such systems include those using thelac repressor from E. coli as a transcription modulator to regulatetranscription from lac operator-bearing mammalian cell promoters (Brown,M. et al., Cell, 49:603-612 (1987)), those using the tetracyclinerepressor (tetR) (Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950(1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)). Other systems include FK506 dimer, VP16 or p65 using astradiol,RU486, diphenol murislerone or rapamycin. Inducible systems areavailable from Invitrogen, Clontech and Ariad. Systems using a repressorwith the operon can be used in the invention. Regulation of transgeneexpression in target cells represents a critical aspect of gene therapy.For example, the lac repressor from Escherichia coli can function as atranscriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters (M. Brown et al., Cell,49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551 (1992)) combined the tetracycline repressor(tetR) with the transcription activator (VP16) to create atetR-mammalian cell transcription activator fusion protein, tTa(tetR-VP16), with the tetO-bearing minimal promoter derived from thehuman cytomegalovirus (hCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.Yao and colleagues (F. Yao et al., Human Gene Therapy, supra)demonstrated that the tetracycline repressor (tetR) alone, rather thanthe tetR-mammalian cell transcription factor fusion derivatives canfunction as potent trans-modulator to regulate gene expression inmammalian cells when the tetracycline operator is properly positioneddownstream for the TATA element of the CMVIE promoter. One particularadvantage of this tetracycline inducible switch is that it does notrequire the use of a tetracycline repressor-mammalian cellstransactivator or repressor fusion protein, which in some instances canbe toxic to cells (M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)), to achieve its regulatable effects.

The effectiveness of some inducible promoters increases over time. Insuch cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g. TetR linked to a TetR byan IRES. Alternatively, one can wait at least 3 days before screeningfor the desired function. While some silencing may occur, it isminimized given the large number of cells being used, for example if atleast 1×10⁴ to about 1×10⁷ cells are used, the effect of silencing isminimal. One can enhance expression of desired proteins by known meansto enhance the effectiveness of this system. For example, using theWoodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).See Loeb, V. E., et al., Human Gene Therapy 10:2295-2305 (1999);Zufferey, R., et al., J. of Virol. 73:2886-2892 (1999); Donello, J. E.,et al., J. of Virol. 72:5085-5092 (1998).

Examples of polyadenylation signals useful to practice the presentinvention include but are not limited to human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

The exogenous genetic material that includes the transgene operablylinked to the regulatory elements may remain present in the cell as afunctioning cytoplasmic molecule, a functioning episomal molecule or itmay integrate into the cell's chromosomal DNA. Exogenous geneticmaterial may be introduced into cells where it remains as separategenetic material in the form of a plasmid. Alternatively, linear DNA,which can integrate into the chromosome, may be introduced into thecell. When introducing DNA into the cell, reagents, which promote DNAintegration into chromosomes, may be added. DNA sequences, which areuseful to promote integration, may also be included in the DNA molecule.Alternatively, RNA may be introduced into the cell.

Selectable markers can be used to monitor uptake of the desired geneinto the hematopoietic stem cells of the invention. These marker genescan be under the control of any promoter or an inducible promoter. Theseare well known in the art and include genes that change the sensitivityof a cell to a stimulus such as a nutrient, an antibiotic, etc. Genesinclude those for neo, puro, and tk, multiple drug resistance (MDR),etc. Other genes express proteins that can readily be screened for suchas green fluorescent protein (GFP), blue fluorescent protein (BFP),luciferase, and LacZ.

The HSC cells can be transduced with a therapeutic gene. For example,the transduction can be via a viral vector such as a retroviral vector(e.g. as described in for example, WO 94/29438, WO 97/21824 and WO97/21825) or a pox viral vector. When transduction is ex vivo, thetransduced cells are subsequently administered to the recipient. Thus,the invention encompasses treatment of diseases amenable to genetransfer into HSCs, by administering the gene ex vivo or in vivo by themethods disclosed herein. For example, diseases including, but notlimited to, β thalassemia, sickle cell anemia, adenosine deaminasedeficiency, recombinase deficiency, recombinase regulatory genedeficiency, etc. can be corrected by introduction of a therapeutic gene.Other indications of gene therapy are introduction of drug resistancegenes to enable normal stem cells to have an advantage and be subject toselective pressure during chemotherapy. Suitable drug resistance genesinclude, but are not limited to, the gene encoding the multidrugresistance (MDR) protein.

Diseases other than those associated with hematopoietic cells can alsobe treated by genetic modification, where the disease is related to thelack of a particular secreted product including, but not limited to,hormones, enzymes, interferons, growth factors, or the like. Byemploying an appropriate regulatory initiation region, inducibleproduction of the deficient protein can be achieved, so that productionof the protein will parallel natural production, even though productionwill be in a different cell type from the cell type that normallyproduces such protein. It is also possible to insert a ribozyme,antisense or other message to inhibit particular gene products orsusceptibility to diseases, particularly hematolymphotropic diseases.

As used herein, therapeutic gene can be an entire gene or only thefunctionally active fragment of the gene capable of compensating for thedeficiency in the patient that arises from the defective endogenousgene. Therapeutic gene also encompasses antisense oligonucleotides orgenes useful for antisense suppression and ribozymes forribozyme-mediated therapy. Therapeutic genes that encode dominantinhibitory oligonucleotides and peptides as well as genes that encoderegulatory proteins and oligonucleotides also are encompassed by thisinvention. Generally, gene therapy will involve the transfer of a singletherapeutic gene although more than one gene may be necessary for thetreatment of particular diseases. The therapeutic gene can be a normal,i.e. wild-type, copy of the defective gene or a functional homolog. In aseparate embodiment, the therapeutic gene is a dominant inhibitingmutant of the wild-type. More than one gene can be administered pervector or alternatively, more than one gene can be delivered usingseveral compatible vectors. Depending on the genetic defect, thetherapeutic gene can include the regulatory and untranslated sequences.For gene therapy in human patients, the therapeutic gene will generallybe of human origin although genes from other closely related speciesthat exhibit high homology and biologically identical or equivalentfunction in humans may be used, if the gene product does not induce anadverse immune reaction in the recipient. For example, a primate insulingene whose gene product is capable of converting glucose to glycogen inhumans would be considered a functional equivalent of the human gene.The therapeutic gene suitable for use in treatment will vary with thedisease. For example, a suitable therapeutic gene for treating sicklecell anemia is a normal copy of the globin gene. A suitable therapeuticgene for treating SCID is the normal ADA gene.

General Considerations

For each of the embodiments described herein, a number of variations canbe made as described below, without departing from the scope of theinvention.

The term “about” in the context of numerical values and ranges refers tovalues or ranges that approximate or are close to the recited values orranges such that the invention can perform as intended, such as having adesired rate, amount, degree or extent of stem cell activity, as isapparent from the teachings contained herein. Thus, this termencompasses values beyond those simply resulting from systematic error.

Angiopoietin-like protein (Angptl) can be any member of a family ofsecreted glycosylated proteins that are similar in structure toangiopoietins (Oike et al., Int. J. Hematol. 80:21-8 (2004)). Similar toangiopoietins, angiopoietin-like proteins contain an N-terminalcoiled-coil domain and a C-terminal fibrinogen-like domain. Unlikeangiopoietins, angiopoietin-like proteins do not bind to the tyrosinekinase receptor Tie2. Angiopoietin-like proteins includeangiopoietin-like proteins 2, 3, 4, 5, 6, and 7. Angiopoietin-likeproteins also include microfibrillar-associated glycoprotein 4 (Mfap4),and analogs and equivalents thereof. Angptl2 has been described by Kim,I. et al. J Biol Chem 274, 26523-8 (1999)). In addition,angiopoietin-like proteins are available commercially (R&D Systems,Abnova Corp). Angiopoietin-like proteins 2, 3, 4, 5, 7, and Mfap4 arepreferred.

Exemplary angiopoietin-like proteins are provided, for example inGenBank as Accession Number AAH12368 (human Angptl2 precursor; SEQ IDNO: 3) Accession Number AAH58287 (human Angptl3 precursor; SEQ ID NO: 4)Accession Number AAH23647 (human Angptl4; SEQ ID NO: 5) and AccessionNumber AAH49170 (human Angptl5; SEQ ID NO: 6). SEQ ID NOs: 3 through 6are shown in FIG. 15. Other suitable angiopoietin-like proteins share atleast 60% sequence homology with any one of SEQ ID NOs: 4 to 6. In otherembodiments, suitable angiopoietin-like proteins share at least 70% orat least 80% or at least 90% sequence homology with SEQ ID NOs: 4 to 6.An exemplary sequence for Angptl7 is found in GenBank Accession No.AAH01881. An exemplary sequence for Mfap4 is found in GenBank AccessionNo. NP_(—)002395. The exemplary sequences of Angptl proteins having theGenBank Accession Nos. provided above are hereby incorporated byreference. In addition to naturally-occurring Angptl sequences, theskilled artisan will further appreciate that suitable Angptl proteinsinclude those proteins that have changes in the naturally occurringamino acid sequence wherein the altered Angptl sequence retainsfunctional ability of the Angptl protein. Suitable alterations includechanges to or elimination of non-essential amino acid residues as wellas conservative amino acid changes (e.g., replacing an amino acidresidue with an amino acid residue having a similar side chain).

Suitable equivalents for angiopoietin-like protein include proteins andpolypeptides having similar biological activity to these factors aswild-type or purified angiopoietin-like proteins (e.g., recombinantlyproduced). Suitable analogs of angiopoietin-like proteins includefragments retaining the desired activity and related molecules. Onepreferred analog is a fragment of the angiopoietin-like proteincontaining the coiled coil domain. For example, the coiled coil domainof angiopoietin-like protein 2. As shown herein, the coiled coil domainstimulates HSC expansion. Another analog is the fibrinogen-like domain.Fragments of Angptls such as the coiled-coil domain and thefibrinogen-like domain may be easier to express and to purify comparedto full-length protein. Molecules capable of binding the correspondingangiopoietin-like protein receptor and initiating one or more biologicalactions associated with angiopoietin-like protein binding to itsreceptor are also within the scope of the invention.

The angiopoietin-like protein can be naturally produced or can beproduced by expressing a gene encoding the angiopoietin-like protein ina suitable host using any suitable expression method. The host can be,for example, bacteria, yeast, or cell culture. The cell culture can be,for example, insect cell culture, or mammalian cell culture. Theangiopoietin-like protein can be glycosylated. In another embodiment,the angiopoietin-like protein is glycosylated in the same orsubstantially the same manner as the naturally occurringangiopoietin-like protein.

As described herein, hematopoietic stem cells have the ability todifferentiate into any of several types of blood cells, including redblood cells, white blood cells, including lymphoid cells and myeloidcells. As described herein, HSCs include hematopoietic cells havinglong-term engrafting potential in vivo. Long term engrafting potential(e.g., long term hematopoietic stem cells) can be determined usinganimal models or in vitro models.

Animal models for long-term engrafting potential of candidate humanhematopoietic stem cell populations include the non-obesediabetic/severe combined immunodeficiency mouse (NOD/SCID) model, theSCID-hu bone model (Kyoizumi et al. (1992) Blood 79:1704; Murray et al.(1995) Blood 85(2) 368-378) and the in utero sheep model (Zanjani et al.(1992) J. Clin. Invest. 89:1179). For a review of animal models of humanhematopoiesis, see Srour et al. (1992) J. Hematother. 1:143-153 and thereferences cited therein. An in vitro model for stem cells is thelong-term culture-initiating cell (LTCIC) assay, based on a limitingdilution analysis of the number of clonogenic cells produced in astromal co-culture after 5 to 8 weeks (Sutherland et al. (1990) Proc.Nat'l Acad. Sci. 87:3584-3588). The LTCIC assay has been shown tocorrelate with another commonly used stem cell assay, the cobblestonearea forming cell (CAFC) assay, and with long-term engrafting potentialin vivo (Breems et al. (1994) Leukemia 8:1095).

As used herein, expansion or propagation includes any increase in cellnumber. Expansion includes, for example, an increase in the number ofhematopoietic stem cells over the number of HSCs present in the cellpopulation used to initiate the culture. Expansion can also includeincreased survival of existing cells, such as hematopoietic stem cells.The term survival refers to the ability to continue to remain alive orfunction.

The cell or cells used to inoculate the cell culture may be derived fromany source including bone marrow, both adult and fetal, cytokine orchemotherapy mobilized peripheral blood, fetal liver, bone marrow orumbilical cord blood. Isolated fractions of cells can be used. Forexample, a purified “side population” (SP) cells obtained from bonemarrow or other sources can be used. Other enriched populations of HSCscan also be used. Methods for isolating enriched populations of HSCs areknown to those in the art, e.g. methods for obtaining SP cells aredescribed in Goodell et al., J. Exp. Med. 183, 1797-806 (Apr. 1, 1996).

Separation of stem cells from a cell population can be performed by anynumber of methods, including cell sorters, (e.g., fluorescence activatedcell sorters) magnetic beads, and packed columns. Exemplary of a highlyenriched stem cell population is a population having the CD34⁺Thy⁻1⁺LIN⁻phenotype as described in U.S. Pat. No. 5,061,620. A population of thisphenotype will typically have an average CAFC frequency of approximately1/20 (Murray et al. (1995) supra; Lansdorp et al., J. Exp. Med. 177:1331(1993)). It will be appreciated by those of skill in the art that theenrichment provided in any stem cell population will be dependent bothon the selection criteria used as well as the purity achieved by thegiven selection techniques. Methods for isolating highly enrichedpopulations of hematopoietic stem cells are further provided in U.S.patent application Ser. No. 5,681,559. The population of cells can befrom any suitable animal. The cells can be derived from a mammal.Suitable mammals include human, non-human primate, cow, horse, dog, cat,mouse and the like. In an embodiment, the cells are human cells, instill another embodiment, the cells are murine cells.

The method of the invention can be used to stimulate the expansion ofany stem cells which expand in the presence of an angiopoietin-likeprotein, including other types of adult stem cells such as endothelialprogenitor cells (Shi, Q. et al. (1998), Blood. 92, 362-367), bonemarrow stromal stem cells (Owen, M. (1988), J. Cell Science Supp. 10,63-76), mesenchymal stem cells (Pittenger, M. F. and Marshak, D. R.(2001), Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds. ColdSpring Harbor, New York: Cold Spring Harbor Laboratory Press 349-374),and skeletal muscle stem cells (Gussoni, E., et al. (1999), Nature. 401,390-394), embryonic stem cells, as well as others. In anotherembodiment, the stem cells are endothelial progenitor cells, which arebelieved to share the same precursor—hemangioblasts—as HSCs.

As used herein, the term cytokine refers to any one of the numerousfactors that exert a variety of effects on cells, for example, inducinggrowth or proliferation or preventing cell death. Non-limiting examplesof additional cytokines which may be used in combination in the practiceof the present invention include, interleukin-2 (IL-2), interleukin 3(IL-3), interleukin 6 (IL-6) including soluble IL-6 receptor,interleukin 12 (IL12), G-CSF, granulocyte-macrophage colony stimulatingfactor (GM-CSF), interleukin 1 alpha (IL-1α), interleukin 11 (IL-11),MIP-1α, leukemia inhibitory factor (LIF), and flt3 ligand. The presentinvention also includes culture conditions in which one or more cytokineis specifically excluded from the medium. Cytokines are commerciallyavailable from several vendors such as, for example, Amgen (ThousandOaks, Calif.), R & D Systems (Minneapolis, Minn.) and Immunex (Seattle,Wash.).

Cytokines useful to promote expansion of hematopoeitic stem cells inmethods of the invention include fibroblast growth factor (FGF) (e.g.FGF-1 or FGF-2), insulin growth factor (e.g. IGF-2, or IGF-1)thrombopoietin (TPO), and stem cell factor (SCF). Accordingly, inanother embodiment, the media includes at least two of FGF, IGF, TPO andSCF or analogs and equivalents thereof. Equivalents thereof includemolecules having similar biological activity to these factors (i.e. FGF,TPO, IGF and SCF) as wild-type or purified cytokines (e.g.,recombinantly produced). Analogs include fragments retaining the desiredactivity and related molecules. For example, TPO is a ligand of the mp1receptor, thus molecules capable of binding the mp1 receptor andinitiating one or more biological actions associated with TPO binding tomp1 are also within the scope of the invention. An example of a TPOmimetic is found in Cwirla et. al., Science 276:1696 (1997).

Culturing includes incubating cells in a suitable medium in vitro. It isunderstood that the descendants of a cell grown in culture may not becompletely identical (either morphologically, genetically, orphenotypically) to the parent cell. Suitable media that can be used forculturing HSCs are known to those in the art. Illustrative media includeHEPES, Dulbecco's MEM, IMDM RPMI-1640, and StemSpan (Stem CellTechnologies) that can be supplemented with a variety of differentnutrients, heparin, antibiotics, growth factors, cytokines, etc.Suitable conditions comprise culturing at 33° to 39° C., and preferablyaround 37° C. The effect of oxygen concentration on culturing HSCs isknown in the art, for example, see Cipolleschi et al. (1993), Blood82:2031-7. HSCs can be cultured in an oxygen concentration of 1 to 10%.In another embodiment, the oxygen concentration is 1 to 5%, for example,1% oxygen. In another embodiment, the HSCs are cultured under hypoxicconditions. Media can be replaced throughout the culture period. Inanother embodiment, half of the medium is replaced twice per week withfresh media. The cells can be cultured from 3 to 30 days. In anotherembodiment, the population of cells including HSCs is cultured for atleast four weeks. In another embodiment, the population of cellsincluding HSCs is cultured for up to two weeks. In another embodiment,the population of cells including HSCs is cultured for 7 to 14 days. Inanother embodiment, the population of cells including HSCs is culturedfor 10 days.

An effective amount is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofthe angiopoietin-like protein and/or cytokine used herein is an amountthat is sufficient to promote expansion of hematopoietic stem cells. Inanother embodiment, an effective amount of the various cytokinesindividually may be from about 0.1 ng/mL to about 500 ng/mL. In anotherembodiment, from about 1 ng/mL to about 200 ng/mL can be used. Inanother embodiment, from about 10 ng/ml to 100 ng/ml of the cytokinescan be used.

An isolated or purified population of cells is substantially free ofcells and materials with which it is associated in nature. For example,isolated hematopoietic stem cells comprise a population of cells that isat least 50% hematopoietic stem cells, or at least 70% hematopoieticstem cells, or at least 80% hematopoietic stem cells. In yet anotherembodiment, an isolated or purified population of hematopoietic stemcells comprises at least 90% hematopoietic stem cells, or is 100%hematopoietic stem cells. Substantially free of stromal cells cancomprise a cell population which, when placed in a culture system asdescribed herein, does not form an adherent cell layer.

A subject or individual is a vertebrate. In an embodiment, theindividual is a mammal. Mammals include, but are not limited to, humans,non-human primates, mice, cows, horses, dogs, cats and the like. In apreferred embodiment, the mammal is a human.

The expanded hematopoietic cells of the invention can be geneticallymodified. As used herein, a genetic modification can include anyaddition, deletion or disruption to the nucleotide sequence of a cell.The methods of this invention are intended to encompass any method ofgene transfer into hematopoietic stem cells, including but not limitedto viral mediated gene transfer, liposome mediated transfer,transformation, transfection and transduction, e.g., viral mediated genetransfer such as the use of vectors based on DNA viruses such asadenovirus, adeno-associated virus and herpes virus, as well asretroviral based vectors. Other non limiting examples of vectors includenon-viral vectors such as DNA/liposome complexes, and targeted viralprotein DNA complexes. To enhance delivery of non viral vectors to acell, the nucleic acid or proteins can be conjugated to antibodies orbinding fragments thereof which bind cell surface antigens, e.g.,Sca-1⁺, IGF2-hFC⁺, and CD31⁺. Where freshly isolated HSCs are used,additional suitable surfaced markers include endoglin⁻ and CD150⁺.Liposomes that also comprise a targeting antibody or fragment thereofcan be used in the methods of this invention.

As used herein, the terms transgene, heterologous gene, exogenousgenetic material, exogenous gene, and nucleotide sequence encoding thegene are used interchangeably and are meant to refer to genomic DNA,cDNA, synthetic DNA and RNA, mRNA and antisense DNA and RNA which isintroduced into the hematopoietic stem cell. The exogenous geneticmaterial may be heterologous or an additional copy or copies of geneticmaterial normally found in the individual or animal. When cells are tobe used as a component of a pharmaceutical composition in a method fortreating human diseases, conditions or disorders, the exogenous geneticmaterial that is used to transform the cells may also encode proteinsselected as therapeutics used to treat the individual and/or to make thecells more amenable to transplantation.

A viral vector is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors such as lentiviral vectors; adenovirusvectors; adeno-associated virus vectors and the like. In aspects wheregene transfer is mediated by a retroviral vector, a vector constructrefers to the polynucleotide comprising the retroviral genome or partthereof, and a therapeutic gene.

The following example is not intended to limit the present invention inany way.

EXAMPLE Materials and Methods

Animals

C57 BL/6 CD45.2 and CD45.1 mice were purchased from the JacksonLaboratory or the National Cancer Institute and were maintained at theWhitehead Institute animal facility. All animal experiments wereperformed with the approval of M.I.T. Committee on Animal Care.

FACS Sorting

Donor bone marrow cells were isolated from 8 to 10 week old C57BL/6CD45.2 mice. To sort SP Sca-1⁺ CD45⁺ cells, adult mouse bone marrow SPcells (stained as previously described (Zhang, C. C. & Lodish, H. F.Blood 103, 2513-21 (2004) and Zhang, C. C. & Lodish, H. F. Blood 105,4314-20 (2005)) were further stained with anti-Sca-1-PE andanti-CD45-FITC followed by cell sorting on a MoFlo® sorter.

To sort cells that bind or that do not bind to Angptl2, 1×10⁶ bonemarrow cells were resuspended in 1 ml conditioned medium, containing ˜1μg/ml FLAG-Angptl2 or FLAG-hFc-Angptl2 as determined by Westernblotting, at 4° C. for 30 min, followed by staining with APC-anti-FLAGM2 or anti-hFc IgG1-PE antibody, respectively. The conditioned mediumfrom mock-transfected cells was used as control.

DNA Array Experiments and Analyses

Total RNA and cRNA were prepared for hybridization to Affymetrix U74Bv2and U74Cv2 mouse chips according to the manufacturer's instructions.Briefly, total RNA was isolated with TRIzol. 15 μg of total RNA was usedfor first strand and second strand cDNA synthesis, followed by in vitrotranscription using Ambion T7 MegaScript Kit to produce biotinylatedcRNAs. Fragmented cRNAs were then hybridized to Affymetrix U74Bv2 andU74Cv2 mouse chips at 45° C. for 16 h. Arrays were washed, stained, andscanned. Microarray data was analyzed by Microarray Suite. Backgroundelements which were not detected in the array of fetal liver CD3⁺ cellsamples (defined as perfect match hybridization not significantlydifferent from mismatch control signal intensity according to MicroarraySuite analysis) were filtered out. An arbitrary raw value of 50 wasallocated for genes whose expression levels were undetectable or whosescan readouts were below 50 in arrays of splenic CD3⁺ or fetal liverGr-1⁺ cell samples, in order to facilitate calculation of the foldchanges. Array measurements for all samples were then normalized witharrays hybridized with cRNAs prepared from the control cells by usingthe median of the hybridization signals of all genes. Transcripts infetal liver CD3⁺ cells that had a normalized value >2.0 were selected.For each candidate transcript, its refseq nucleotide ID was retrievedfrom the Affymetrix data center, followed by transfer into the Refseqprotein sequence ID. The protein sequence was then obtained using batchentrez from the NCBI Entrez database. The signal peptides were predictedusing the SignalP web server (on the world wide web atcbs.dtu.dk/services/SignalP/), based on both Neural networks and HiddenMarkov models (Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G.Protein Eng 10, 1-6 (1997), and Bendtsen, J. D., Nielsen, H., vonHeijne, G. & Brunak, S. J Mol Biol 340, 783-95 (2004)). Candidateproteins with signal peptides expressed in fetal liver CD3⁺ cells at alevel greater than two-fold that of adult CD3⁺ and fetal liver Gr-1⁺cells were selected for further study.

Production of Coiled-Coil Domain and Fibrinogen-Like Domain of Angptl2

The DNA encoding human Angptl2 fused with FLAG peptide sequence at the cterminus was constructed as Flag-Angptl2. Human Angptl2 and Pro 100-Lys330 of human IgG1 Fc sequences were linked by a DNA sequence encodingIEGRMD linker peptide (SEQ ID NO.: 1) to form Angptl2-hFc. The wholefragment was inserted into pcDNA3.1, downstream of CMV promoter. Theplasmid was transfected into 293T cells using lipofectamine 2000(Invitrogen) and conditioned medium at 48 hour after transfection wascollected. The conditioned medium with about 1 μg/ml of IGF-2-hFcdetermined by western blotting was used in the subsequent staining ofbone marrow cells.

Similar to constructing FLAG-hAngptl2, a cDNA encoding the coiled-coildomain or fibrinogen-like domain of human Angptl2 fragments wasgenerated. The former contained hAngptl2 amino acids Arg 1-Lys 249 andthe latter Arg 1-Pro 76 fused to Arg 276-His 493. Both were fused at theC-terminus with a FLAG epitope. FLAG tagged human Fgl1 or human Mfap4were also constructed with a FLAG fused at the C-terminus of the encodedproteins. These plasmids were transfected into 293T cells and theconditioned medium were collected similar to the procedure for producingFLAG-Angptl2.

Western Blots

Purified proteins or crude proteins in conditioned medium were analyzedby electrophoresis on 4 to 12% NuPage Bis-Tris polyacrylamide gels(Invitrogen), and proteins were electroblotted onto nitrocellulosemembranes. The membranes were probed with the horseradishperoxidase-conjugated anti-FLAG M2 monoclonal antibody (Sigma; at 1:5000dilution) or a combination of human Angptl2-specific primary monoclonalantibodies (in FIG. 1: 1 μg/ml of clone 239829; in FIG. 15A: 1 μg/ml of239809.11, 239813.111, 239816.111, 239819.111, 239829.111, 239830.11,239833.11, 239834.11, and 239835.111, all gifts from R&D Systems),incubated with the horseradish peroxidase-conjugated goat-anti-mouseantibody (at 1:2000 dilution) and detected by chemiluminescence with theECL kit (Amersham, Arlington Heights, Ill.).

Real Time PCR

Total RNA was isolated from indicated fetal liver or bone marrow cellpopulations. First-strand cDNA was synthesized using SuperScript II RT(Invitrogen). Samples were analyzed in triplicate 25 μl reactions (300nM of primers, 12.5 μl of Master mix), which was adapted from thestandard protocol provided in SyBR Green PCR Master Mix and RT-PCRProtocol provided by Applied Biosystems. Primers were purchased fromQiagen (QT00151179 for Angptl2 and QT00110824 for Angptl3). The defaultPCR protocol was used on an Applied Biosystems Prism 7000 SequenceDetection System. The mRNA level of Angptl2 and Angptl3 in eachpopulation was normalized to the level of 18S RNA transcripts present inthe same sample.

Angiopoietin-Like Proteins

FLAG-hAngptl2, the coiled-coil domain of hAngptl2, FLAG-taggedfibrinogen-like domain of hAngptl2, FLAG-hAngptl4, FLAG-hFgl1, andFLAG-hMfap4, were all produced by transient transfection of 293T cellsusing Lipofectamine 2000 (Invitrogen). After transfection, the cellswere cultured overnight in IMDM with 10% FBS, and then washed with IMDMbefore being cultured in serum-free StemSpan medium (StemCellTechnology) for another 24 h. The conditioned medium was harvested andused in experiments in FIGS. 1, 2, and 5 b. Medium from mock-transfectedcells was always used as a negative control. Serum-free conditionedmedium cultured mock-transfected 293T cells for 4 h before addition ofpurified Angptl2 or Angptl3 was used in the experiments in FIG. 3. Topurify FLAG-Angptl2 and FLAG-Angptl4, the correspondingplasmid-transfected 293T cells were cultured in IMDM with 10% FBS for 48h or 72 h and the conditioned medium was collected for anti-FLAGaffinity purification.

Purified mouse angiopoietin-like protein 3 (mAngptl3) was produced insf21 cells using a baculovirus expression system, and was a gift fromR&D Systems. GST-hAngptl5, a fusion protein of GST and humanangiopoietin-like protein 5 (hAngptl5) and produced by a cell-free wheatgerm in vitro transcription/translation system, was purchased fromAbnova Corporation, Taiwan. Bacterially-expressed hAngptl2 and hAngptl7were gifts from R&D Systems.

Production of Tagged-Angptl2 and other FLAG-Tagged Proteins

The cDNA encoding human Angptl2 (Kim, I. et al. J Biol Chem 274, 26523-8(1999)) fused with a FLAG peptide sequence (as FLAG-hAngptl2) or withPro 100-Lys 330 of human IgG1 Fc sequence followed by FLAG (asFLAG-hFc-hAngptl2) at the C-terminus was constructed. The DNA wasinserted into the pcDNA3.1 (−) vector (Invitrogen) downstream of the CMVpromoter. Plasmids were transfected into 293T cells using lipofectamine2000 (Invitrogen) and the serum-containing conditioned medium wascollected at 48 h and 72 h after transfection.

Purification of FLAG-Angptl2 and FLAG-Angptl4

Serum-free conditioned medium as detailed above was harvested after48-72 h from FLAG-hAngptl2 or FLAG-hAngptl4 transfected 293T cells. Onetablet/50 ml of the Complete Protease Inhibitor Cocktail (Roche), 5μg/ml PMSF, and 100 mM NaCl were added, and the medium was applied to ananti-FLAG epitope immunoaffinity column (Anti-FLAG M2 affinity Gel,Sigma), using 500 μl of resin per 500 ml of conditioned medium. Thecolumn was subsequently washed 10 times with a total of 100 volumes ofTBS (50 mM Tris, pH 7.4, 150 mM NaCl) and the FLAG-hAngptl2 orFLAG-hAngptl4 was eluted with 0.1 mg/ml FLAG peptide (N-DYKDDDDK-C; SEQID NO.: 2) dissolved in TBS.

Cell Culture

Twenty BM SP Sca-1⁺ CD45⁺ cells isolated from 6 to 9 or 8 to 10 week oldC57BL/6 CD45.2 mice were plated in 100 or 160 μl of StemSpan serum-freemedium (StemCell Technologies) supplemented with 10 μg/ml heparin(Sigma), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2(all from R&D Systems), and 10 ng/ml human FGF-1 (Invitrogen), with orwithout the indicated amounts of angiopoietin-like proteins. In someexperiments, the cells were plated in one well of a U-bottom 96-wellplate (3799; Corning). As indicated in the individual experiments anddetailed above, some of these proteins had been purified, and otherswere added from the conditioned medium of transfected 293T cells. Mediumfrom mock-transfected cells was always used as a negative control. After3 to 10 d of culture as indicated, the cells were harvested forcompetitive transplantation. Unconditioned serum-free mediumsupplemented with 10 μg/ml heparin, 10 ng/ml mouse SCF, 20 ng/ml mouseTPO, 20 ng/ml mouse IGF-2, and 10 ng/ml human FGF-1 is termedunconditioned STIF medium, and the conditioned medium collected from293T cells which was then supplemented with the above cytokines istermed conditioned STIF medium. For the purpose of competitivetransplantation, cells from at least 6 culture wells were pooled andmixed with competitor cells before the indicated numbers of cells weretransplanted into each mouse.

Competitive Reconstitution Analysis

The indicated numbers of CD45.2 donor cells were mixed with 1×10⁵ or2×10⁵ freshly isolated CD45.1 competitor bone marrow cells, and themixture injected intravenously via the retro-orbital route into each ofa group of 6 to 9 week old CD45.1 mice previously irradiated with atotal dose of 10 Gy. To measure reconstitution of transplanted mice,peripheral blood was collected at the indicated times post-transplantand the presence of CD45.1⁺ and CD45.2⁺ cells in lymphoid and myeloidcompartments were measured as described (Zhang, C. C. & Lodish, H. F.Blood 103, 2513-21 (2004) and Zhang, C. C. & Lodish, H. F. Blood 105,4314-20 (2005)). Briefly, peripheral blood cells were collected byretro-orbital bleeding, followed by lysis of red blood cells andstaining with anti-CD45.2-FITC, and anti-CD45.1-PE, or anti-Thy1.2-PE(for T-lymphoid lineage), anti-B220-PE (for B-lymphoid lineage),anti-Mac-1-PE, anti-Gr-1-PE (cells costaining with anti-Mac-1 andanti-Gr-1 were deemed the myeloid lineage), or anti-Ter119-PE (forerythroid lineage) monoclonal antibodies (BD Pharmingen). The “Percentrepopulation” shown in all Figures except FIGS. 1 c and 1 d was based onthe staining results of anti-CD45.2-FITC and anti-CD45.1-PE. In allcases FACS analysis of the above listed lineages was also performed toconfirm multilineage reconstitution. Calculation of CRUs in limitingdilution experiments was conducted using L-Calc software (StemCellTechnologies) (Zhang, C. C. & Lodish, H. F. Blood 105, 4314-20 (2005)).

Primary Human Cells

Primary human cord blood cells were purchased from Cambrex (Poietics™Mononuclear cells from Human Cord Blood, Cat #2C-150A). Cells wereplated at 10⁶ cells/ml of StemSpan serum-free medium (StemCellTechnologies) supplemented with 10 μg/ml heparin (Sigma), 10 ng/ml mouseSCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2 (all from R&D Systems),and 10 ng/ml human FGF-1 (Invitrogen), and with 100 ng/ml Angptl3 orAngptl5. Medium volume was increased by adding fresh medium at day 5, 8,12, and 15 to maintain cell densities at 5×10⁵ to 1.5×10⁶ cells/ml.

Transplant into NOD/SCID Mice

Cultured progeny from human mononuclear cord blood cells were collectedat the indicated days and injected into sub-lethally irradiated (350rad) NOD-SCID mice (purchased from Jackson). Two months aftertransplantation, bone marrow nucleated cells from transplanted animalswere analyzed by flow cytometry for the presence of human CD45⁺ cells.Mice were considered to be positive for human HSC engraftment when atleast 0.1% CD45⁻ human cells were detected among mouse bone marrowcells.

Results

Fetal Liver CD3⁺ Cells Express Angptl2 and Angptl3

Secreted or membrane proteins that are specifically expressed in E15mouse fetal liver CD3⁺ cells but not in two cell populations—adult CD3⁺cells and fetal liver Gr-1⁺ cells—that do not support HSC maintenance orexpansion in culture were identified by microarray analysis (Table 1).

TABLE 1 FLCD3⁺ SpnCD3⁺ FLGr-1⁺ Fold Fold (raw) (raw) (raw) (1)/(2)(1)/(3) Name GI NP (1) (2) (3) (Normalized) (Normalized) claudin 1310048432 NP_065250 4184.2 26.6 10.7 96.5 96.5 RIKEN cDNA 21313426NP_084345 1774.3 21.2 8.7 42.3 42.3 4432416J03 chemokine 12963823NP_076274 936.2 18.7 8.0 20.7 20.7 (C—X—C motif) ligand 7angiopoietin-like 31560520 NP_036053 583.2 4.2 5.8 13.8 13.8 2 popeyedomain 11612497 NP_071713 581.6 4.5 3.7 9.5 9.5 containing 2 expressed31981988 NP_705768 470.0 3.2 3.8 9.3 9.3 sequence C85492 RIKEN cDNA34304038 NP_899082 375.1 7.5 3.3 8.0 8.0 2210020M01 QIL 1 protein23346595 NP_694792 281.5 2.2 2.2 5.8 5.8 RIKEN cDNA 22122497 NP_666132277.8 3.8 3.5 4.7 4.7 C730027E14 RIKEN cDNA 28077025 NP_083209 237.7 3.14.1 5.3 5.3 1300010M03 RIKEN cDNA 28077031 NP_083166 214.4 2.3 2.6 3.83.8 4931414P19 claudin 14 34328494 NP_062373 208.5 2.9 26.4 4.8 4.8chemokine (C—C 9625035 NP_062523 201.1 1.9 37.1 3.2 3.2 motif) ligand 24RIKEN cDNA 27370416 NP_766508 182.0 2.6 2.9 4.3 4.3 1100001I19angiopoietin-like 33469117 NP_038941 171.6 2.0 15.5 3.3 3.3 3 neuritin 123956286 NP_705757 60.2 1.8 3.9 3.0 3.0

Both angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3)were found to be secreted proteins specifically expressed in this stemcell supportive population (Table 1, FIG. 11A). These proteins are alsofound to be expressed in adult bone marrow (BM) cells including the SPCD45⁺ Sca-1⁺ highly enriched HSC population (FIG. 11B and FIG. 11C).These two proteins have not previously been implicated in HSC biology.

Freshly Isolated and 4 Day Cultured Bone Marrow HSCs Bind to Angptl2

Fusion proteins of Angptl2 with the human IgG Fc fragment or FLAGpeptide were generated. When these fusion proteins were used to isolatepopulations of freshly isolated or cultured bone marrow cells that canor cannot bind Angptl2, and followed by reconstitution analysis of them,it was found that Angptl2 is capable of binding to the majority offreshly isolated BM HSCs (FIG. 12A), and to all 4 day cultured HSCs(FIG. 12B). This demonstrates that a receptor for Angptl2 is expressedon most freshly isolated HSCs and all cultured HSCs.

In the experiment shown in FIG. 12A, freshly isolated adult CD45.2 bonemarrow cells were incubated at 4° C. for 30 min with conditioned mediumfrom control transfected 293T cells or cells transfected with theAngptl2-hFc—expression vector; the latter medium contained 1 μg/mlAngptl2-hFc. Cells were then stained with anti-human IgG1-PE. On average12.8% of total bone marrow cells bind to Angptl2-hFc and 5.3% of 4 daycultured bone marrow cells bind to Angptl2. 5×10⁴ positively stainedcells and the same number of negatively stained cells were transplantedtogether with 2×10⁵ CD45.1 competitor cells into lethally irradiatedCD45.1 mice (n=4-5). Peripheral blood cells were analyzed for thepresence of CD45.2⁺ cells in lymphoid and myeloid compartments at 3weeks and 4 months after transplant.

In the experiment shown in FIG. 12B, adult CD45.2 bone marrow cells werecultured in serum-free medium with SCF, TPO, IGF-2, and FGF-1 asdescribed (Reya, T. et al. Nature 423, 409-14 (2003)) for 4 days. Thecells were then stained with Flag-Angptl2—containing medium (˜1 μg/mlFlag-Angptl2) as the same as described in FIG. 3. 5.3% of total cellsbind to Angiopoietin-FLAG. 1300 positively stained cells and 8000negatively stained cells were transplanted together with 2×10⁵ CD45.1competitor cells into lethally irradiated CD45.1 mice (n=4). Peripheralblood cells were analyzed for the presence of CD45.2⁺ cells in lymphoidand myeloid compartments at 3 weeks and 3 months after transplant.

Angptl2 and Angptl3 Stimulate Ex Vivo Expansion of HSCs

A plasmid containing the entire coding sequence for human Angptl2 with aFLAG tag fused at the C-terminus in the eukaryotic expression vectorpcDNA3.1(−) (FLAG-Angptl2) was constructed. Following transienttransfection of 293T cells, the culture supernatant contained secretedFLAG-Angptl2 migrating with the expected ˜60 kD size (FIG. 1). As shownherein, the majority of freshly isolated LT-HSCs and all LT-HSCscultured for 4 d bound this hormone (FIG. 12).

In the experiment shown in FIG. 2, twenty CD45.2 bone marrow SP Sca-1⁺CD45⁺ cells were seeded into each well of a 96-well U-bottom plate, inserum-free conditioned medium collected from mock transfected 293T cells(S/F: serum-free medium, SCF: serum-free medium with 10 ng/ml SCF, S+T:serum-free medium with 10 ng/ml SCF and 20 ng/ml TPO, S+T+I+F:serum-free medium with 10 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, and10 ng/ml FGF-1), or in serum-free conditioned medium from 293T cellstransfected with Flag-Angptl2 (S/F+Angptl2: serum-free medium with ˜100ng/ml Angptl2, S+A: serum-free medium with 10 ng/ml SCF and ˜100 ng/mlAngptl2, S+T+A: serum-free medium with 10 ng/ml SCF, 20 ng/ml TPO, and˜100 ng/ml Angptl2, S+T+I+F+A: serum-free medium with 10 ng/ml SCF, 20ng/ml TPO, 20 ng/ml IGF-2, 10 ng/ml FGF-1, and ˜100 ng/ml Angptl2).Images shown were taken after 3 days of culture, under a phase-contrastmicroscope.

A representative of two independent experiments (FIG. 3A) shows thatAngptl2 is a stimulator of ex vivo expansion of LT-HSCs. In this studyAngptl2 was not purified but was contained in the conditioned medium of293T cells transfected with a FLAG-Angptl2 expression vector;conditioned medium from mock-transfected cells served as a negativecontrol. When 20 adult BM SP CD45⁺ Sca-1⁻ cells, a highly enriched HSCpopulation (Camargo, F. D., Green, R., Capetanaki, Y., Jackson, K. A. &Goodell, M. A. Nat Med 9, 1520-7 (2003)), were cultured for 5 d inserum-free medium supplemented with SCF, essentially all long-term(LT)-HSC activity was lost, measured by competitive reconstitution (FIG.3A, compare bars 7 and 12 to bars 6 and 11, respectively). After culturein the same medium with SCF and 100 ng/ml FLAG-Angptl2 for 5 d, LT-HSCactivity was dramatically increased (compare bars 8 and 13 to bars 7 and12, respectively). Similarly, HSCs cultured for 10 d in the presence ofSCF, TPO, IGF-2, FGF-1 and Angptl2 achieved a tremendous increase ofLT-HSC activity compared to culture in the same medium without Angptl2(compare bars 10 and 15 to bars 9 and 14, respectively). Stem cellscultured in the presence of Angptl2 repopulated both lymphoid andmyeloid lineages of the primary recipients at 9 months post-transplant(FIG. 3B) as well as in secondary transplanted mice (FIG. 3C),indicating a net expansion of LT-HSCs. After 9 months followingtransplants all mice were healthy and no tumors were observed. Additionof 100 ng/ml FLAG-Angptl2 also caused an increase in expansion of ST-HSCactivity, measured at 3 weeks post-transplant (compare bar 3 with 2, and5 with 4).

Importantly, culturing highly enriched HSCs in serum-free mediumcontaining SCF, TPO, IGF-2, and FGF-1 was found to result in an 8-foldincrease of LT-HSC numbers (Zhang, C. C. & Lodish, H. F. Blood 105,4314-20 (2005)). An additional increase in the extent of HSC expansionby adding Angptl2 was observed. Therefore, Angptl2 is a novel growthfactor for HSCs, whose effect is synergistic with other HSC growthfactors.

To isolate pure recombinant Angptl2, conditioned medium fromFLAG-Angptl2 transfected 293T cells was collected and the FLAG-taggedprotein was purified by immunoaffinity chromatography using animmobilized monoclonal antibody specific for the FLAG epitope. SDS-PAGEof the eluted fraction showed two major bands, one at the positionexpected for the full-length FLAG-Angptl2 (˜60 kDa), and the other asmaller peptide of ˜36 kDa (FIG. 4A, lane 3). The mammalian-expressedfull-length FLAG-Angptl2 had a higher molecular weight thanbacterially-expressed Angptl2 (compare lane 3 to lane 2), consistentwith a previous result that mammalian-expressed Angptl2 is glycosylated(Kim, I. et al. J Biol Chem 274, 26523-8 (1999)). Western blotting withan anti-FLAG M2 antibody, which recognizes the C-terminal FLAG epitope,stained both bands (FIG. 4B) as did an Angptl2-specific monoclonalantibody (FIG. 4C). Thus the FLAG-Angptl2 underwent partial proteolysisduring purification.

As shown in FIG. 5, purified mammalian-expressed Angptl2 stimulated theproliferation of hematopoietic stem cells. Twenty CD45.2 bone marrow SPSca-1⁺ CD45⁺ cells were cultured for 10 days in serum-free medium with10 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1, or inthe same medium with 100 ng/ml purified Flag-Angptl2 (as in FIG. 4).Then the cells were cotransplanted with 1×10⁵ CD45.1 competitors intoCD45.1 recipients (n=5). Engraftment 3 weeks (left panel) and 2 months(right panel) post-transplant were shown.

The limiting dilution competitive repopulation assay in FIG. 7 showsthat culture of purified HSCs with Angptl2 or Angptl3, together withother growth factors, results in a greater than 20-fold expansion ofLT-HSC numbers. The frequency of long-term repopulating cells (CRU) infreshly isolated BM SP CD45⁺ Sca-1⁺ cells is 1 per 23 at 3 monthspost-transplant (95% confidence interval for mean: 1/15 to 1/35, n=25;FIG. 7A, bottom line) or 1 in 39 at 6 months post-transplant (95%confidence interval for mean: 1/24 to 1/63; FIG. 7A, top line). That is,as calculated from Poisson statistics, injection of on average of 23 or39 freshly isolated BM SP CD45⁺ Sca-1⁻ cells is sufficient to repopulate63% (=1-1/e) of transplanted mice. After the cells were cultured for 10d in serum-free conditioned STIF medium with Angptl2, the number ofcells was too few to be counted reliably. However, based on the numberof cells initially added to the culture, the CRU of the cultured cellswas 1/1.1 at 3 months post-transplant (FIG. 7B, bottom line; 95%confidence interval for mean: 1/0.5 to 1/2.3, n=30) or 1/1.6 at 6 monthspost-transplant (FIG. 7B, top line; 95% confidence interval for mean:1/1.1 to 1/2.3). In other words, injection of the cultured progeny ofonly 1.1 or 1.6 freshly isolated BM SP CD45⁺ Sca-1⁺ cells is sufficientto repopulate 63% of the mice. Thus the data in FIG. 7B shows that thenumber of LT-HSCs (6 months post-transplant) increases 24 fold (=39/1.6)after culture.

The same strategy was used to measure the effect of purified Angptl3.The CRU of the cultured cells was 1/0.7 at 3 months post-transplant(FIG. 7C, bottom line; 95% confidence interval for mean: 1/0.3 to 1/1.7,n=24) or 1/1.3 at 6 months post-transplant (FIG. 7C, top line; 95%confidence interval for mean: 1/0.9 to 1/2.0), again relative to thenumber of cells initially added to the culture. Therefore, culture of BMSP CD45⁺ Sca-1⁺ cells in the presence of purified Angptl3 for 10 dresults in a 30 (=39/1.3) fold expansion of repopulating LT-HSCs (6months post-transplant).

Expansion of HSC activity by Angptl3, like that by Angptl2, was highlyreproducible. In two additional experiments, progenies of 20 BM SP CD45⁺Sca-1⁺ cells after 10 d of culture in serum-free conditioned STIF mediumwith 100 ng/ml Angptl3 showed 65.3%±4.0% and 73.1%±3.1% (n=5) ofengraftments respectively at 4 months post-transplant. Thus the culturesystem consistently achieved dramatic increases of the repopulationactivities of HSCs. FIG. 6 shows that mammalian cell-specificposttranslational modifications of Angptl2 facilitate its stimulation ofex vivo HSC expansion. Confirming the result in FIG. 3A, addition of 100ng/ml mammalian-expressed Angptl2 significantly increased HSC activityafter culture (FIG. 6, compare bars 3 and 8 to bars 1 and 6,respectively). By contrast, 100 ng/ml bacterially-expressed Angptl2 wasunable to stimulate HSC expansion over STIF medium alone (FIG. 6,compare bars 2 and 7 to bars 1 and 6, respectively). This suggests thatsome mammalian-specific modification, presumably glycosylation (see FIG.4A, lanes 2 and 3), positively correlates with the ability of Angptl2 tostimulate LT-HSC expansion. The coiled-coil domain of Angptl2 alsostimulated ex vivo HSC expansion (right panel of FIG. 6, compare bars 4and 9 to bars 1 and 6, respectively).

Several Angptl Family Members Stimulate HSC Expansion.

Angptl2 and Angptl3 belong to a family of angiopoietin-like proteins(Oike, Y., Yasunaga, K. & Suda, T. Int J Hematol 80, 21-8 (2004)). FIG.5 shows that several members of this family, like Angptl2 and Angptl3,are capable of stimulating HSC expansion in culture. The effects ofpurified Angptl3, Angptl5, or Angptl7, as well as the coiled-coil domainof Angptl2, Angptl4, or Microfibrillar-associated glycoprotein 4 (Mfap4)in 293T conditioned medium were tested for stimulation of ex vivoexpansion of HSCs. All these factors support the increase of HSCactivity after culture.

As shown in FIG. 8, Angptl2-coiled coil domain, Angptl4, and Mfap4expressed in 293T cell conditioned medium stimulated the proliferationof hematopoietic stem cells. Twenty CD45.2 bone marrow SP Sca-1⁺ CD45⁺cells were cultured for 5 days in serum-free mock transfectedconditioned medium with 10 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, 10ng/ml FGF-1 (lanes 1 and 8), in conditioned medium with the same factorsand full-length Angptl2 (lanes 2 and 9), Angptl2 coiled-coil domain(lanes 3 and 10), Angptl2 fibrinogen-like domain (lanes 4 and 11),Angptl4 (lanes 5 and 12), Fibrinogen-like 1 (lanes 6 and 13), orMicrofibril-associated glycoprotein 4 (lanes 7 and 14). The cells werethen cotransplanted with 1×10⁵ CD45.1 competitors into CD45.1 recipients(n=5). Engraftment 2 weeks (left panel) and 1 month (right panel)post-transplant are shown.

FLAG-tagged Angptl4 was generated by transient transfection of 293Tcells followed by immunoaffinity purification using an immobilizedanti-FLAG monoclonal antibody. In addition, purified Angptl3 (producedin sf21 cells using a baculovirus system), GST-fused Angptl5 (producedby a cell-free wheat germ in vitro transcription/translational system),and Angptl7 (produced by a bacterial expression system) (left panel,FIG. 10A) were obtained.

Bone marrow SP Sca-1⁺ CD45⁺ cells were cultured for 5 d in serum-freeunconditioned STIF medium, in the presence of 100 ng/ml of Angptl3,Angptl4, Angptl5, or 1 μg/ml of Angptl7 (FIG. 10A). Addition of Angptl3to the culture stimulated both ST-HSC and LT-HSC expansion (FIG. 10A,compare bars 2, 7, and 12 to bars 1, 6, and 11, respectively). Asignificant increase of both ST- and LT-HSC activities was also observedafter culture with Angptl5, and also 1 μg/ml of bacterially-expressedAngptl7 (compare bars 1, 6, and 11 to bars 4, 9, and 14, as well as bars5, 10, and 15, respectively). Angptl 4 stimulated at least some HSCactivity when tested at 2 weeks and 1 month post transplant (FIG. 9).However, 100 ng/ml Angptl4 was ineffective in stimulating HSC expansionat 3 months and 6 months (FIG. 10, compare bars 3, 8, and 13).

The effects of two orthologs of Angptls, microfibrillar-associatedglycoprotein 4 (Mfap4) (Zhao, Z. et al. Hum Mol Genet 4, 589-97 (1995))and fibrinogen-like 1 (Fgl1) (Yamamoto, T. et al. Biochem Biophys ResCommun 193, 681-7 (1993)) were also tested. Both full-length proteinswere FLAG-tagged and generated by transient transfection of 293T cells.They were secreted into the medium and detected by Western blotting(FIG. 10B, left panel). Both ˜100 ng/ml FLAG-Mfap4 and FLAG-Fgl1 wereapplied to HSCs directly in the serum-free conditioned STIF medium (FIG.10B). Competitive reconstitution analysis demonstrated that Mfap4 didstimulate ex vivo expansion of BM SP Sca-1⁺ CD45⁺ LT-HSCs after a 5 dculture whilst Fgl1 did not (FIG. 10B).

Angptl 5 Stimulates HSC Activity in Human Cord Blood Cells

As demonstrated in FIG. 13, culturing primary human cord blood cells inSTIF medium supplemented with Angptl5 resulted in an approximately 3fold increase in frequency of SCID mouse repopulating activity.

In conclusion, angiopoietin-like proteins are shown to be importantnovel growth factors for HSCs, including human HSCs. The stimulation ofex vivo expansion of HSCs by angiopoietin-like proteins most likelyresults from a direct effect of the hormone on these cells. As describedherein, that the majority of freshly isolated LT-HSCs and all LT-HSCscultured for 4 d bound Angptl2; thus the unknown receptor(s) of Angptl2is expressed on cultured HSCs. Furthermore, the cultures describedherein contained only 20 highly enriched HSCs in 160 μl of medium.Because of this low cell density, it is unlikely that any accessorycell(s) in this population respond to Angptl2 by producing sufficientamounts of other growth factors to stimulate HSC expansion.

A simple serum-free culture system for bone marrow HSCs using saturatinglevels of SCF, TPO, IGF-2, and FGF-1 was developed; during 10 d ofculture of highly enriched HSCs an 8-fold increase in numbers of LT-HSCswas observed. (Zhang, C. C. & Lodish, H. F. Blood 105, 4314-20 (2005)).SCF, IGF-2, and FGF-1 all activate receptor protein-tyrosine kinases,whilst TPO signals through a member of the cytokine receptor superfamilythat requires a Janus Kinase to activate intracellular signaltransduction pathways. As demonstrated herein, addition of any ofseveral members of the Angptl family—specifically Angptl2, Angptl3,Angptl, 4, Angptl5, and Angptl7, as well as Mfap4—result in a furtherincrease in HSC activities. This suggests that the Angptls activatesignal transduction pathways in addition to those activated by SCF, TPO,IGF-2, or FGF-1. As demonstrated herein, at least Angptl2 and Angptl3are produced by HSC supportive mouse fetal liver CD3⁺ cells; therefore,both Angptl2 and Angptl3 may normally function in vivo to stimulateexpansion of fetal liver, and perhaps also adult, HSCs. As demonstratedby FIG. 13, Angptl5 stimulates human HSCs, demonstrating that Angptlsare also an important growth factor family for human HSCs. Therefore,Angptls are useful for ex vivo expansion of HSCs. In addition, thesefactors are useful for ex vivo expansion of these cells as part of anHSC transplantation or gene therapy protocol.

It is to be understood that the description, specific examples and data,while indicating exemplary embodiments, are given by way of illustrationand are not intended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the discussion, disclosure and data containedherein, and thus are considered part of the invention.

1-38. (canceled)
 39. A kit for propagating hematopoietic stem cells(HSCs) in vitro comprising: a) medium suitable for culturing one or morecells comprising an HSC, b) at least one angiopoietin-like protein, andc) at least one additional growth factor selected from the groupconsisting of insulin-like growth factor (IGF), fibroblast growth factor(FGF), thrombopoietin (TPO), and stem cell factor (SCF). 40-46.(canceled)
 47. The kit of claim 39, wherein the angiopoietin-likeprotein is selected from the group consisting of angiopoietin-likeprotein 2, angiopoietin-like protein 3, angiopoietin-like protein 4,angiopoietin-like protein 5, angiopoietin-like protein 7, and Mfap4. 48.The kit of claim 39, wherein when in use, the angiopoietin-like proteinis present in the medium at a concentration of about 0.1 ng/mL to about500 ng/mL.
 49. The kit of claim 39, wherein the angiopoietin-likeprotein is a recombinant protein.
 50. The kit of claim 39, wherein thekit comprises SCF.
 51. The kit of claim 39, wherein when in use, theadditional growth factor is present in the medium at a concentration ofabout 0.1 ng/mL to about 500 ng/mL.
 52. The kit of claim 39, wherein theone or more cells are selected from the group consisting of bone marrowcells, umbilical cord blood cells, and fetal liver cells.
 53. The kit ofclaim 39, wherein the one or more cells are human cells.
 54. The kit ofclaim 39, wherein the one or more cells are primary cells.
 55. The kitof claim 39, further comprising an instruction to culture the one ormore cells for at least five days.
 56. A kit for propagatinghematopoietic stem cells (HSCs) in vitro comprising: a) medium suitablefor culturing primary cells comprising an HSC; b) at least oneangiopoietin-like protein selected from the group consisting ofangiopoietin-like protein 2, angiopoietin-like protein 3,angiopoietin-like protein 4, angiopoietin-like protein 5,angiopoietin-like protein 7, and Mfap4; and c) at least one additionalgrowth factor selected from the group consisting of insulin-like growthfactor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), andstem cell factor (SCF).
 57. The kit of claim 56, wherein when in use,the angiopoietin-like protein is present in the medium at aconcentration of about 0.1 ng/mL to about 500 ng/mL
 58. The kit of claim56, wherein the angiopoietin-like protein is a recombinant protein. 59.The kit of claim 56, wherein the kit comprises SCF.
 60. The kit of claim56, wherein when in use, the additional growth factor is present in themedium at a concentration of about 0.1 ng/mL to about 500 ng/mL
 61. Thekit of claim 56, wherein the primary cells are selected from the groupconsisting of bone marrow cells, umbilical cord blood cells, and fetalliver cells.
 62. The kit of claim 56, wherein the primary cells arehuman cells.
 63. The kit of claim 56, further comprising an instructionto culture the one or more cells for at least five days.
 64. A method ofpropagating hematopoietic stem cells (HSCs) in vitro comprising: (a)providing the kit of claim 39; and (b) culturing the one or more cellsin the medium comprising the angiopoietin-like protein and theadditional growth factor.
 65. A method of propagating hematopoietic stemcells (HSCs) in vitro comprising: (a) providing the kit of claim 56; and(b) culturing the primary cells in the medium comprising theangiopoietin-like protein and the additional growth factor.